Chemical Sensor Enhanced by Direct Coupling of Redox Enzyme to Conductive Surface

ABSTRACT

The invention provides a method, apparatus and system for detecting electrochemical oxidoreduction activity mediated by a redox enzyme at a site remote from the enzyme. In one embodiment, the method comprises immobilizing the redox enzyme on a first region of a conductive surface and contacting a substrate capable of producing a detectable signal upon oxidation or reduction with a second region of the conductive surface. The second region is electrically coupled with the first region and the redox enzyme is not present in the second region. The method further comprises exposing the immobilized redox enzyme to conditions that effect oxidation or reduction of the enzyme, and detecting oxidation or reduction of the substrate at the second region. The invention can be adapted for detecting a plurality of analytes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 11/364,436,filed Feb. 28, 2006, now U.S. Pat. No. ______, the entire contents ofwhich are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The invention disclosed herein was made with Government support underGrant No. 5U01-DE014971-04, awarded by the National Institutes ofHealth. The government has certain rights in this invention.

TECHNICAL FIELD OF THE INVENTION

This invention relates generally to the manufacture of sensors andmethods of using same. The invention amplifies chemical sensormeasurements by coupling a redox enzyme to a conductive surface. Theinvention exploits direct electron transfer to achieve detection remotefrom the site of the redox enzyme.

BACKGROUND OF THE INVENTION

The well-understood catalytic cycle between horseradish peroxidase (HRP)and its commonly utilized colorimetric substrate, tetramethylbenzidine(TMB), proceeds as follows. First, HRP undergoes a two-electronoxidation of its ferriheme prosthetic group by hydrogen peroxide insolution. The oxidized HRP, known as compound-I, consists of anoxyferryl iron and a porphyrin τ cation radical [Everse, J., et al.,Peroxidases in Chemistry and Biology. Vol. 1, 1991, Boca Raton: CFCPress. 620]. In the next reaction, the oxidized HRP converts the TMB inbulk solution from its normal reduced state to a colored oxidized form.Specifically, in a first step, a one-electron oxidation of TMB resultsin the formation of a cation radical intermediate. In a second reaction,the cation radical is further oxidized to form a yellow colored diimine.Two of the intermediate cation radicals may also combine to form a bluecharge-transfer complex, which may be readily quantified withspectrophotometry [Josephy, P. D., et at. Journal of BiologicalChemistry, 1982. 257(7): p. 3669-3675; Bally, R. W. and T. C. J.Gribnau, J Clin Chem Clin Biochem, 1989. 27(10): p. 791-796]. In thepresence of dextran sulfate or other precipitating agents, the normallysoluble oxidized form of TMB reacts to form an insoluble dark bluecolored precipitate [McKimm-Breschkin, J. L., Journal of ImmunologicalMethods, 1990. 135: p. 277-280]. As a result of the TMB oxidation steps,the enzyme is reduced to its native resting state, and may then bere-oxidized by hydrogen peroxide to restart the cycle. This peroxidasereaction sequence is well known in the literature and has been used fora number of detection schemes [Volpe, G., et al., Analyst, 1998. 123: p.1303-1307; Alfonta, L., et at., Analytical Chemistry, 2001. 73(21): p.5287-5295; Loo, R. W., et al., Analytical Biochemistry, 2005. 337(2): p.338-342].

Direct electron transfer (DET) between an electrode material andredox-active biomolecules was first reported in 1977, and involved theuse of cyclic voltammetry measurements of cytochrome-c etectrochemistry[Eddowes, M. J. and H. A. G. Hill, Journal of the ChemicalSociety—Chemical Communications, 1977. 21: p. 771-772; Yeh, P. and T.Kuwana, Chemistry Letters, 1977. 10: p. 1145-48]. Since these initialreports, efficient direct electron transfer has been documented for anumber of redox enzymes, the majority of which contain a metallocenter,namely a heme group [Habermuller, L., et al. Fresenius Journal ofAnalytical Chemistry, 2000. 366(6-7): p. 560-568; Gorton, L., et al.,Analytica Chimica Acta, 1999. 400: p. 91-108]. DET has also beenestablished between HRP and a number of electrode surfaces, includingcarbon and graphite materials, gold, and platinum [Ruzgas, T., et at.,Journal of Electroanalytical Chemistry, 1995, 391: p. 41-49]. HRP is oneof the most commonly studied redox enzymes for coupling electrontransport directly to conductive surfaces [Ruzgas, T., et al., Journalof Electroanalytical Chemistry, 1995. 391: p. 41-49; Yaropolov, A. I.,et al. Bioelectrochemistry and Bioenergetics, 1978. 5(1): p. 18-24;Ferapontova, E., Electroanalysis, 2004. 16: p. 1101-1112]. It is knownthat this coupling can be used to monitor redox reactions mediated byHRP, such as those involved in biosensor applications, by monitoringcurrent flow to an electrode onto which the HRP is adsorbed [Ghindilis,A. L., et al. Electroanalysis, 1997. 9(9): p. 661-674.].

There remains a need, however, for improved and more flexible methods ofamplifying redox reactions used in chemical sensors. The inventiondisclosed herein addresses these needs and others by providing a meansof exploiting electron transfer to remote sites via electrically coupledconductive surfaces.

SUMMARY OF THE INVENTION

The invention provides a method for detecting electrochemicaloxidoreduction activity mediated by a redox enzyme at a site remote fromthe enzyme. In one embodiment, the method comprises immobilizing theredox enzyme on a first region of a conductive surface and contacting asubstrate that produces a detectable signal upon oxidation or reductionwith at least one additional region of the conductive surface. Theadditional region is electrically coupled with the first region and theredox enzyme is not present in the additional region. The method furthercomprises exposing the immobilized redox enzyme to conditions thateffect oxidation or reduction of the enzyme, and detecting oxidation orreduction of the substrate at the additional region. Examples of asubstrate capable of producing a detectable signal upon oxidation orreduction include a colorimetric or fluorogenic enzyme substrate, suchas tetramethylbenzidine (TMB), 4-chloro-1-naphthol (4-CN) and3,3′-diaminobenzidine (DAB) or dihydroxyphenoxazine (Amplex® Red). Insome embodiments, the substrate produces the detectable signal viareaction with an agent and the detectable signal comprises acolorimetric, fluorogenic or precipitating product, such as wherein thesubstrate is tetramethylbenzidine and the agent is dextran sulfate.

Typically, the redox enzyme comprises horseradish peroxidase, glucoseoxidase, alcohol oxidase, lactate oxidase, choline oxidase, cholesteroloxidase, glutmateoxidase or amino acid oxidase. In one example, theconditions that effect oxidation of the redox enzyme comprise contactwith hydrogen peroxide. The conductive surface typically comprises ametal film, such as gold, silver, copper, platinum or aluminum film, orcarbon or graphite materials.

The electrochemical oxidoreduction activity can be indicative of thepresence of an analyte, thereby providing a method for analytedetection. The methods of the invention can be performed using flow orstopped flow conditions. Both the substrate and the analyte can beintroduced using flow or stopped flow conditions.

Also provided is an apparatus and system for detecting electrochemicaloxidoreduction activity mediated by a redox enzyme at a site remote fromthe enzyme. The system comprises an apparatus having a conductivesurface that has a first region and at least one additional region, anda means for detecting oxidation or reduction of a substrate thatproduces a detectable signal upon oxidation or reduction at theadditional region. In this embodiment, a redox enzyme is immobilized onthe first region of the conductive surface, the additional region iselectrically coupled with the first region, and the redox enzyme is notpresent in the additional region.

The invention further provides a method, apparatus and system fordetecting a plurality of analytes. The method comprises contacting asolution suspected of containing a plurality of analytes with aplurality of electrically coupled paired regions or sets of electricallycoupled regions. Each of the electrically coupled sets of regions iselectrically isolated from the other electrically coupled regions and afirst region of each of the sets of regions comprises an immobilizedredox enzyme that is oxidized or reduced, directly or indirectly, uponcontact with an analyte. At least one additional region of each set ofregions is in contact with a substrate that produces a detectable signalupon oxidation or reduction. The method further comprises exposing theimmobilized redox enzymes to conditions that effect oxidation orreduction of the enzymes, and detecting oxidation or reduction of thesubstrates at the additional region(s) of each set of regions.

Also provided is a sensor for detecting a plurality of analytes. Thesensor comprises a plurality of electrically coupled paired regions orsets of electrically coupled regions, wherein each paired region iselectrically isolated from the other paired regions. A first region ofeach of the paired regions comprises an immobilized redox enzyme that isoxidized or reduced upon contact with an analyte, and at least oneadditional region of each of the paired regions is electrically coupledwith the first region, and the redox enzyme is not present in theadditional region. Upon contact with a substrate that produces adetectable signal upon oxidation or reduction, oxidation or reduction ofthe substrate is detected in the additional region(s).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic overview of the surface electrochemistry systemutilized in the examples described herein, including surface-boundenzymes, a bulk substrate solution, and the resultant precipitationreaction.

FIG. 2. Surface plasmon resonance (SPR) image of the pump-controlled HRPrinse-out procedure with PBS, with flow from left-to-right. The arrowcorresponds to the general lateral position of the patterned enzymeregions. Note that excess HRP in the original patterned regions of thegold surface is deposited downstream of the original patterns afterrinsing with PBS, i.e., there is no HRP on the gold surface upstream ofor lateral to the original patterns.

FIG. 3. SPR images throughout the addition of TMB and a subsequent rinsewith PBS. Flow is from left-to-right in all images. The numberscorrespond to image number, where images were captured every 2 seconds.Note that the TMB precipitate rapidly appears in regions without HRP,e.g., upstream of the location of the original HRP patterns.

FIG. 4. SPR data that indicates precipitate formation at sites remotefrom the HRP requires a conductive path between the regions of interest.The precipitate forms in the bare gold region upstream of andelectrically couple to the patterned HRP. However, the precipitate doesnot form in the isolated bare gold region upstream of the patterned HRP.Again, flow is from left to right, and images were captured every 2seconds.

FIG. 5. SPR data that indicates precipitate formation at sites remotefrom the HRP only requires a conductive path between the regions ofinterest (given HRP redox activity at the initial site and a TMBprecipitating substrate solution at the remote site). Specifically, acontinuous volume of TMB (or some other component of the bulk substratesolutions between the regions of interest is not required. Sequence ofSPR images with TMB in both the upper and lower fluid streams. Five HRPregions were patterned in the upper stream, and PBS was introduced intothe center stream. Note the extensive precipitate formation in bothTMB-containing streams, including the lower stream, which contained noenzyme. Again, flow is from left to right, and images were capturedevery 2 seconds. The image numbers were 31, 33, 37, 90, 160, and 200,from left to right, top to bottom.

FIG. 6. Photograph of the final sample described in FIG. 5 above. Goodagreement was observed between regions of high SPR intensity and regionsof precipitate formation. Note that the SPR instrument used in this workforeshortens the image in the x-dimension.

FIG. 7. Design for a multi-analyte assay. The 3×5 array of rectangularregions (and the two large rectangular regions on either side of thearray) were coated with gold. Each region of the array, since it iselectrically isolated from the other regions, may be treated as anindependent measurement.

FIG. 8. Representative SPR image of the array format before theformation and spreading of the precipitate.

FIG. 9. Representative SPR image of the array format after the formationand spreading of the precipitate. The signal in each of the regions isindependent.

FIG. 10. Schematic of a basic embodiment of the invention, where themethod is utilized as a sensitive hydrogen peroxide detector.

FIG. 11. Schematic of a competitive immunoassay variation of the presentinvention.

FIG. 12. Integrated scheme of the present invention, involving a secondenzyme that generates hydrogen peroxide in proportion to the amount ofbivalent sample antigen present.

FIG. 13. Schematic illustration of an apparatus of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based on the discovery of a novel method of applyingthe process of direct electron transfer between surface-bound redoxmolecules and an electrical conductor to the enhancement of chemicaldetection. The invention relates to direct coupling between a redoxenzyme (e.g., horseradish peroxidase, HRP) and a conductive surface as amethod of amplifying chemical sensor measurements. The invention isbased on a surprising effect of detectable and measurable precipitateformation at sites remote from the redox enzyme that is mediated byelectrical coupling through a continuous conductive surface between thesites. This discovery provides for significant amplification of theoriginal signal at sites remote from the original site as well as at theoriginal site. The invention also provides for parallel detection ofmultiple samples and/or multiple analytes by taking advantage of theability to electrically isolate different electrochemical reactions.

In general, the method involves two or more conductive regions (orsurfaces) that are electrically connected. On one conductive region anelectrochemical reaction occurs through any of a number of mechanisms.The electrons gained or lost through this reaction are then transportedto or from a second conductive region, where a correspondingelectrochemical reaction occurs. This sequence of electron-shuttlingreactions occurring at two distinct sites thereby increases the areaover which the electrochemical reaction occurs. The electron transportbetween the aforementioned conductive regions may occur through a numberof mechanisms. The specific oxidation and reduction reactions are alsowide-ranging, and may include enzymatic redox reactions. This method hasutility in a range of electrochemical sensors and also has applicabilityto a variety of detection platforms, including optical detectionmethods.

Definitions

All scientific and technical terms used in this application havemeanings commonly used in the art unless otherwise specified. As used inthis application, the following words or phrases have the meaningsspecified.

As used herein, “redox enzyme” refers to an enzyme having chemicalmoiety capable of undergoing a reduction (acceptance of one or moreelectrons) or oxidation (donation of one or more electrons) between aconductive surface and the enzyme.

As used herein, a “conductive” surface means an electrically conductivesurface.

As used herein, “electrically coupled” refers to two or more regionsthat are electrically coupled with each other.

As used herein, under “flow conditions” refers to a state of continuousliquid solution flow.

As used herein, “stopped flow conditions” refers to a state in whichliquid solution flow is halted, such as to allow for measurement oranalysis.

As used herein, “a” or “an” means at least one, unless clearly indicatedotherwise.

Overview

This novel surface electrochemistry method has been demonstrated usingan enzyme precipitation system, in which the surface-bound redox-activemolecule is the enzyme HRP, the substrate is TMB, and a precipitatingagent having properties similar to dextran sulfate is used. In thisdemonstration, the conductor was a thin uniform gold film of finite areadeposited on an electrical insulator. Note that in this case the goldlayer was not electrically coupled to a measuring system.

One of the primary requirements for efficient electron transfer betweenimmobilized redox molecules and an electrode is a short, unobstructedpath between the molecule's active site and the electrode surface[Habermuller, L., et al. Fresenius Journal of Analytical Chemistry,2000. 366(6-7): p. 560-568]. This requirement can be met by directadsorption of the molecule to the electrode. If this requirement is notmet, precipitate formation is localized only to the regions containingHRP. This was demonstrated in recent work involving an enzymeprecipitate amplification scheme on a self-assembled monolayer-coatedgold surface. In this setup, the redox molecule (HRP) was elevated abovethe electrode surface, and strict localization of the biocatalyzedprecipitate to regions containing HRP was observed [Pyo, H. B., et al.,Langmuir, 2005. 21(1): p. 166-171].

Methods

In brief, the present invention involves a method for increasing thearea over which an electrochemical reaction occurs following directelectron transfer (DET) between immobilized redox-active molecules and aconductive surface. In a typical embodiment, the redox-active moleculeis the enzyme HRP; the electrochemical reactions are a sequence ofoxidation and reduction reactions occurring between hydrogen peroxide,HRP, and TMB; the conductive surface is a thin gold film, and thedetection method is surface plasmon resonance imaging.

The exemplary method focuses on two conductive regions, denoted zone Aand zone B, that are in electrical communication with each other. Onzone A, HRP is immobilized in such a way as to allow direct coupling tothe gold firm through its heme prosthetic group. To begin the process,the surface-bound HRP molecules are oxidized by the addition of hydrogenperoxide to the buffer surrounding the enzymes. The gold film in zone A,which is in electrical contact with the HRP prosthetic group, thentransfers electrons to re-reduce the HRP; the gold firm in zone B (thatdoes not contain HRP) is now biased positive in proportion to the rateof oxidation of the HRP molecules. TMB at the gold film in zone B isthen oxidized and thereby converted to an insoluble precipitate at asite remote from the HRP. The novel aspect of this phenomenon is thatany conducting region in electrical contact with zone A, even at aremote distance, can be the site of oxidation and precipitation of TMBas long as the HRP at zone A is re-reduced.

The accumulation of the detectable precipitate, which otherwise islimited by the fact that it covers the HRP and prevents influx ofchemicals to the immobilized enzyme, is thereby greatly increased viathis method. The formation of the insoluble enzyme precipitate on thegold surface may be readily detected qualitatively by eye (the productis dark blue), or quantitatively by optical absorbance, ellipsometry, orSPR-based techniques. We observed rapid signal amplification inelectrically coupled remote regions not containing HRP, as well as inthe region containing HRP.

The primary application of this phenomenon is the detection of chemicalsthat either directly or indirectly initiate the oxidation or reductionof the immobilized redox molecule. In the above general description ofthe invention, the initial reaction may involve a variety ofredox-active molecules. The only requirement is that electrons aretransferred during the reaction. In addition to direct electrontransfer, the electrons may be shuttled to the electrode surface througha variety of mediators [Gorton, L., et al., Analytica Chimica Acta,1999. 400: p. 91-108]. Likewise, the electrode material may be anyconductive material, including noble metals and carbon nanotubes, aslong as electrons are readily transferable through the material. Theelectrical connection between the initial and final reaction regions maybe continuous, such as in the case of a single gold coating layer, or itmay be a conductive wire that connects the two regions. It is notnecessary to employ a precipitating substrate system. The substratesystem may produce a colorimetric change, such as in the case of asoluble TMB substrate solution, or a fluorescence change such as in thecase of the commercially available Amplex®Red substrate of HRP.

Lastly, the detection and measurement of the electrochemical signal maybe accomplished through any technique capable of measuring electricalcurrent and a variety of other techniques that will depend on thespecific signal generated by the choice of enzyme-substrate system. Fora precipitate-based signal, detection techniques include surface plasmonresonance (SPR)-based techniques (spectroscopy and imaging) andelectrochemical quartz crystal microbalance. Fluorescence detectionmethods would be appropriate for fluorescence-based signals andabsorbance measurement systems would be appropriate for signalsresulting in a colorimetric change.

The method can also be used to detect multiple analytes. In thisembodiment, the sensor comprises a plurality of electrically isolatedconductive regions. At each conductive region, a redox enzyme isdirectly coupled to a portion of the conductive surface. Detection ofeach analyte is amplified and its signal can be detected independentlyfrom the signal from other analytes. Lastly, this method may be appliedunder static or flow conditions.

Apparatus and System

A representative apparatus in accordance with the invention isillustrated schematically in FIG. 13. A conductive surface 10 has afirst region 12 and at least one additional region 14, wherein theregions are electrically coupled via the conductive surface 10. A redoxenzyme 16 is immobilized on the first region 12, but not on the second,or additional region(s) 14. A detectable signal 18 can be produced onboth the first region 12 and at least one additional region 14, althougha signal need not be generated in the first region. Embodiments adaptedfor detection of multiple analytes comprise a plurality of sets ofelectrically coupled regions, wherein each set of electrically coupledregions comprises the elements illustrated in FIG. 13. The plurality ofelectrically coupled regions are electrically isolated from each other,and the first region 12 of each set of regions can be designed to detecta different analyte.

The apparatus can be part of a system for detecting electrochemicaloxidoreduction activity and/or for detection of one or more analytes.The system comprises an apparatus or sensor as described herein and ameans for detecting oxidation or reduction of a substrate capable ofproducing a detectable signal upon oxidation or reduction at theadditional region(s). The detectable signal can be detected by any meansof signal detection known in the art, such as an optical detectionsystem. Examples of detection systems include but are not limited to: aCCD camera, surface plasmon resonance (SPR)-based techniques(spectroscopy or imaging), electrochemical quartz crystal microbalance,and fluorescence or other optical detection, such as is used inELISA-based assays. For some embodiments, the detecting means comprisesSPR-based techniques (spectroscopy or imaging), electrochemical quartzcrystal microbalance, and/or any other technique capable of measuringformation of an insoluble precipitate layer. In addition, a techniquecapable of measuring electrical current can be used.

EXAMPLES

The following examples are presented to illustrate the present inventionand to assist one of ordinary skill in making and using the same. Theexamples are not intended in any way to otherwise limit the scope of theinvention.

Example 1 Lateral Electron Transfer Between Electrically ConnectedRegions

In this example, we employed an established enzyme precipitation systeminvolving the enzyme horseradish peroxidase (HRP), and its colorimetricsubstrate, 3,3′,5,5′-tetramethylbenzidine (TMB). In brief, the specificenzymatic catalytic cycle is as follows. HRP is first oxidized byhydrogen peroxide, the oxidized HRP then, in turn, oxidizes a moleculeof TMB into an insoluble blue form that, upon reaction with aprecipitating agent, results in precipitate formation; the reduced HRPis subsequently re-oxidized by hydrogen peroxide, and the cycle canbegin again [Everse, J., et al., Peroxidases in Chemistry and Biology,Vol. 1. 1991, Boca Raton: CFC Press. 620]. The general experimentalprotocol in the initial demonstration involved HRP directly adsorbed toa gold surface, such that electrons may be transferred between theconductive surface and the enzyme's heme prosthetic group [Ruzgas, T.,et al., Journal of Electroanalytical Chemistry, 1995. 391: p. 41-49;Yaropolov, A. I., et al. Bioelectrochemistry and Bioenergetics, 1978.5(1) p. 18-24; Ferapontova, E., Electroanalysis, 2004. 16: p.1101-1112]. Upon addition of TMB, electrons are transferred betweenelectrically connected gold regions, and the substrate is rapidlyconverted to an insoluble precipitate at sites remote from thesurface-immobilized HRP molecules. The precipitate formation is detectedby surface plasmon resonance imaging (SPRI) or optical absorption oflight reflected by the gold surface. This phenomenon greatly increasesthe area over which the electrochemical reaction occurs and thusamplifies the original signal. The primary application of this method isthe detection of chemicals that either directly or indirectly affect theoxidation or reduction of redox molecules immobilized on a conductivesurface.

These experiments show that the electrochemical precipitate spreadingprocess is rapid and significant. FIG. 1 schematically depicts thesystem used to investigate this method. The enzyme used in thisinvestigation was horseradish peroxidase (Sigma-Aldrich, St. Louis, Mo.,USA) in phosphate buffered saline (PBS) and the substrate solution was3,3′,5,5′-tetramethylbenzidine (United States Biological, Swampscott,Mass., USA) in an acetate buffer containing hydrogen peroxide andprecipitating agents. The first step of the demonstration involvedpatterning the enzyme solution directly onto a gold-coated microscopeslide using a piezoelectric inkjet printing system (MicroFabTechnologies, Plano, Tex., USA). Thus, as required by theelectrochemistry process, a redox-active molecule was placed in closeproximity to a conductive surface.

For most experiments, a microfluidic flow cell was then assembled fromlaminate sheets of Mylar® (Fralock, Santa Clara, Calif., USA) andadhesive, and the pre-patterned gold-coated microscope slide was used asthe bottom layer of the flow cell. The assembled microfluidic flow cellwas then fitted into a manifold on a custom-built SPR microscope [Fu,E., et al., Review of Scientific Instruments, 2004. 75(7): p. 2300-2304;Fu, E., et al. Review of Scientific Instruments, 2003. 74(6): p.3182-3184] and rinsed with PBS to remove any free and/or looselyadsorbed HRP molecules. A controlled, laminar flow rinse was performedto ensure that the excess HRP would only be deposited downstream of thepatterned regions.

After establishing baseline SPR images with PBS as the bulk fluid, theTMB solution was pumped through the microfluidic flow cell at a rate of1 μl/s through the use of syringe pumps (Kloehn, Las Vegas, Nev. USA).The sensor surface was monitored via the SPR microscope throughout thecourse of the electrochemical and enzyme precipitation reactions.Finally, the TMB solution was rinsed from the sensing cell using PBS andthe signal amplification and electrochemical spreading due to theprecipitate formation was examined both qualitatively and quantitativelyusing SPR imaging.

SPR images of the HRP rinse-out procedure, such as that in FIG. 2, whereflow was in the left-to-right configuration, suggested that transport ofany excess enzyme in the patterned regions was restricted to thedownstream regions of the laminar flow rinse (right side of the patternsin the image).

The time sequence following the introduction of TMB to the SPR sensingcell is illustrated in the SPR images in FIG. 3. In all images, flow wasin the left-to-right orientation. The numbers in the lower-right cornerscorrespond to the image number, which were captured every two seconds.The three dark rectangular regions correspond to the patterned enzymeareas. The final image depicts the gold surface following extensiverinsing with PBS. Note that the precipitate remained after rinsing andwas neither restricted to the patterned regions nor discrete regions tothe right of the patterned patch where excess HRP may have adsorbedduring the initial rinse. The precipitate either formed at or becamelocalized to regions that contained no enzyme (both upstream of andlateral to the original enzyme patches), suggesting extensive electrontransfer throughout the gold surface.

This general experimental protocol was then modified in a subsequentexperiment, where a specially designed Mylar® mask was applied to themicroscope slides during the gold deposition process, such that a regionof the slide contained an electrically isolated “island” of gold. Thisconductive region, of dimensions 2 mm by 2 mm, was surrounded on allsides by a 1 mm-wide region of insulating glass. Small regions of 0.1mg/ml HRP were then patterned with the piezoelectric microdispenserdownstream of the isolated gold region. A single-channel microfluidicflow cell was then affixed to the protein patterned slide and the TMBsolution was pumped through the microchannel as before, with flow in theleft-to-right orientation. As in previous experiments, the precipitatewas observed to spread rapidly to regions not containing any enzyme.However, the gold region electrically isolated by the region of glassremained free of the precipitate. This result is depicted in the timesequence of images in FIG. 4, and demonstrates the requirement of anelectrical connection between the HRP region and the region notcontaining HRP, in order to obtain precipitation formation in thelatter.

The electrochemical mechanism involved in this invention disclosure wasfurther exemplified in an experiment involving three microfluidicstreams flowing in parallel in a single microchannel. This three-inletmicrofluidic configuration allowed for the physical separation of twoTMB solution streams, one in contact with immobilized enzyme patterneddirectly on the gold surface and one in contact with a bare gold regionat the opposite wall of the microchannel. Note that this second streamwas not in contact with any enzyme molecules. The center streamcontained only buffer, and the widths of the various streams were variedto control the electron transfer distance.

After patterning the enzyme in discrete rectangular regions, thesubstrates were rinsed thoroughly with PBS under microfluidic flowconditions. As illustrated in the sequence of SPR images in FIG. 5, TMBwas introduced into both the upper (enzyme-containing region) and lower(no enzyme in the region) fluid streams, with the center buffer streamoccupying a relatively small width (˜15%) of the entire microchannel.With TMB already in the lower stream, no precipitate formation wasobserved until TMB was also present in the upper enzyme-containingstream. As indicated in the sequence of images, rapid and significantformation and spreading of the precipitate was then observed in both theupper and lower portions of the channel. This result indicates that theonly requirement for precipitate formation at a site remote from the HRPregion, given HRP activity at the initial site and a TMB precipitatingsubstrate solution at the remote site, is an electrical connectionbetween the regions. Specifically, a continuous volume of TMB (or someother component of the bulk substrate solution) between the regions ofinterest is not required.

This result was verified visually, as presented in FIG. 6, as the blueprecipitate was present along the length of the channel in stripes alongthe upper and lower walls of the channel, i.e. in the same patternobserved in the SPR images.

To explore the utility of the present invention in future quantitativeassays, including those involving the detection of multiple analytes, apreliminary experiment was performed that investigated the effect of arange of enzyme concentrations on the extent of the electrochemistryreaction. It was expected that different enzyme concentrations (and/orperoxide concentrations) would generate different rates of precipitateformation in the regions surrounding the enzyme patterns (i.e. regionsnot containing enzyme). To begin, microscope slides were selectivelypatterned with a thin gold layer, generating fifteen isolated 1 mm×1 mmsquares of conductive gold. The Mylar® mask design used during the golddeposition process is shown in FIG. 7, where regions outlined in bluerepresent regions to be covered with gold. HRP was then patterned ontoeach of the fifteen gold-coated square regions as described above. Theslides received different volumes of 0.01 mg/ml HRP, effectivelyfunctionalizing the gold surfaces with different enzyme concentrations.Patterning with both a low droplet density and a low proteinconcentration ensured sub-monolayer protein coverage. A TMB solution wasthen added to each slide, initiating the electrochemical process, andthe reaction was monitored via SPR microscopy.

FIGS. 8 and 9 depict representative SPR images of a patterned gold slidewith localized enzyme regions before and after the formation andspreading of the precipitate, respectively. In FIG. 9, the enzymeregions appear as the dark rectangular regions within the fifteenrectangles; the higher intensity regions surrounding the patternedenzyme zones represent the precipitate, and the surrounding brighterregions are the bare glass substrate. No visable precipitate formationwas observed on the glass regions. Potential methods of quantificationof the signal include the rate of precipitate formation or an end-pointprecipitate signal measurement. This result also suggests the potentialfor rapid detection and amplification of multiple analytes in parallel.

Example 2 Chemical/Biochemical Detection Schemes Utilizing this NovelDetection Method

This method is generally applicable to any technique involving thedetection of chemicals that either directly or indirectly produce theoxidation or reduction of an immobilized redox molecule. This includes arange of biosensor applications, including those detailed in thefollowing schemes. Note that while the discussion of these schemesincludes specific molecules, reactions, and conductive surfaces to aidin comprehension, the actual schemes should not be limited to thesespecific examples.

Scheme 1. The schematic in FIG. 10 depicts a basic representation of thepresent invention. In this scheme, HRP is immobilized on a goldsubstrate and the addition of hydrogen peroxide followed by TMB (plusprecipitating agents) causes the surface electrochemistry andprecipitation reactions to occur at sites remote from the HRP, asdescribed in detail in previous sections. BSA is added in the secondstep in order to block the surface against non-specific binding. In thiscontext, the present invention may be used as a sensitive hydrogenperoxide sensor.

Scheme 2. A straightforward modification of this first scheme addsextensive utility to the invention. This second class of schemesinvolves the indirect detection of a range of analytes through theconcomitant generation and detection of peroxide. For example, a largenumber of enzyme oxidases exist that, by definition, react withmolecular oxygen to catalyze the oxidation of a substrate. A by-productof this reaction is often hydrogen peroxide, which may be detected as inScheme 1 above.

As a specific example of this scheme, imagine that glucose oxidase iseither immobilized or in bulk solution upstream of a pre-patterned HRPregion on a conductive surface. With the addition of the appropriatesubstrates, in this case, glucose and molecular oxygen, the glucoseoxidase enzyme will produce hydrogen peroxide in proportion to theamount of glucose present. With the exception that hydrogen peroxidewill now be generated internally as part of an additional reaction,steps 3-5 of Scheme 1 will then be followed directly, resulting in asensitive glucose detector. Alternatively, the glucose oxidase moleculesmay be substituted with any of the following examples of oxidizingenzymes to be a sensitive detector for a range of analytes: alcoholoxidase, lactate oxidase, choline oxidase, cholesterol oxidase,glutamate oxidase, and amino acid oxidase [Ruzgas, T., et al., AnalyticaChimica Acta, 1996. 330: p. 123-138].

Scheme 3. Another possible scheme, which integrates a competitiveimmunoassay with the novel surface electrochemistry system, is depictedin FIG. 11. With HRP conjugated to an analyte of interest andimmobilized on the conductive surface, a solution containing thecorresponding antibody and competitor analyte molecules is added to thesystem. Binding of the available antibody molecules (singly ornon-bound) to the surface-immobilized analytes then inactivates thoseHRP molecules, either through stern or chemical inhibition.Alternatively, the antibody may be linked to a specific inhibitor of theenzymatic reaction. Either way, the extent of precipitate formation andspreading is dependent upon the amount of analyte captured.

Scheme 4. The final scheme of the present invention, which is depictedin FIG. 11, is a more complex and integrated version of Scheme 2outlined above. The underlying principle remains the same, whereby theformation of hydrogen peroxide is proportional to the presence ofanother analyte and indirectly generates the electrochemical signalhighlighted in this invention disclosure. Specifically, the hydrogenperoxide, a key initiating component of the TMB precipitation reaction,is generated by an enzyme conjugated to a secondary antibody specific tothe desired analyte of interest. This highly integrated scheme thereforefurther broadens the utility of the invention, such that any bivalentanalyte may be linked to the formation of hydrogen peroxide and theinitiation of this novel electrochemistry reaction. Note that, in theexample below, glucose oxidase is used as a model enzyme label for thesecondary antibody, but any enzyme that produces hydrogen peroxide (oranother molecule capable of oxidizing HRP or another redox enzyme) as aproduct of a chemical reaction could be utilized.

Throughout this application various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to describemore fully the state of the art to which this invention pertains.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A sensor for detecting a plurality of analytes comprising a pluralityof sets of electrically coupled regions, wherein each set of regions iselectrically isolated from the other sets of regions and wherein eachset of regions comprises: (a) a continuous conductive surface having afirst region and at least one additional region, wherein a redox enzymeis immobilized on the first region of the conductive surface, whereinthe additional region is electrically coupled via the continuousconductive surface with the first region and the redox enzyme is notpresent in the additional region; and (b) means for detecting oxidationor reduction of a substrate located at the additional region, whereinthe substrate is capable of producing a detectable signal upon oxidationor reduction at the additional region.
 2. The sensor of claim 1, whereinthe means for detecting oxidation or reduction comprises fluorescence orabsorbance.
 3. The sensor of claim 1, wherein the means for detectingoxidation or reduction comprises surface plasmon resonance.
 4. A methodfor detecting a plurality of analytes comprising: (a) contacting asolution suspected of containing a plurality of analytes with the sensorof claim 1, (b) contacting at least one additional region of each set ofregions with a substrate that produces a detectable signal uponoxidation or reduction; (c) exposing the immobilized redox enzymes toconditions that effect oxidation or reduction of the enzymes; and (d)detecting oxidation or reduction of the substrates at the additionalregion of each set of regions.
 5. The method of claim 4, wherein thecontacting of steps (a) and (b) comprises introducing the solution underflow or stopped flow conditions.
 6. The method of claim 4, wherein thecontacting of steps (a) and (b) is concurrent.
 7. The method of claim 4,wherein the substrate that produces a detectable signal upon oxidationor reduction comprises a colorimetric or fluorogenic enzyme substrate.8. The method of claim 4, wherein the substrate produces the detectablesignal via reaction with an agent and the detectable signal comprises acolorimetric, fluorogenic or precipitating product.
 9. The method ofclaim 4, wherein the redox enzyme comprises horseradish peroxidase,glucose oxidase, alcohol oxidase, lactate oxidase, choline oxidase,cholesterol oxidase, glutamate oxidase or amino acid oxidase.
 10. Themethod of claim 4, wherein the conditions that effect oxidation of theredox enzyme comprise contact with hydrogen peroxide.
 11. The method ofclaim 4, wherein the substrate comprises tetramethylbenzidine (TMB),4-chloro-1-naphthol (4-CN) and 3,3′-diaminobenzidine (DAB) ordihydroxyphenoxazine (AMPLEX® Red).
 12. The method of claim 11, whereinthe substrate is tetramethylbenzidine and the agent is dextran sulfate.13. The method of claim 4, wherein the conductive surface comprises ametal film.
 14. The method of claim 13, wherein the metal comprisesgold, silver, copper, platinum or aluminum.
 15. The method of claim 4,wherein the electrochemical oxidoreduction activity is indicative of thepresence of an analyte.